Investigating effects of aluminum on Saccharomyces cerevisiae:
A model for laboratory-based investigative teaching
INTRODUCTION
Aluminum toxicity has been found to be a major constraint to crop productivity (Anoop, et. al,
2003), a neurotoxic agent in animals (Zatta, 2000) and has been proposed to be a cofactor in
human diseases such as Alzheimer’s, Parkinson’s, anemia, growth disorders, glucose intolerance,
and cardiac arrest (Canada’s Federal Health Department, 2005). According to Barnett, 2000
“mechanisms of toxicity include inhibition of enzyme activity and protein synthesis, alterations in
nucleic acid function, and changes in cell membrane permeability.” In addition, aluminum
exposure alters gene expression in the mitochondria affecting enzymes involved in the glycolysis
pathway (Zatta, 2000). It is unknown exactly how aluminum can affect so many cellular
functions, but it is generally believed that aluminum acts as a competitive inhibitor of several
essential elements including magnesium, calcium, and iron (MacDiarmid and Gardner, 1998). It
has also been suggested that aluminum attacks “cell membrane phospholipids and membrane
proteins and enhances the peroxidation of cell membranes in both animal cells and plants” (Ezaki,
et. al, 1997). Although there have been several studies which examine gene expression of a few
genes in response to aluminum exposure, there is a lack of data on how an entire genome
responds to the presence of aluminum. In this research we used microarray technology to
examine the potential effect of aluminum exposure on gene expression in Saccharomyces
cerevisiae (yeast), a model organism that shares roughly 31% of its genome with humans
(NHGRI, 1996). Additionally this project was used as a pilot study to bring investigative
genomics research tools such as the microarray into the hands of undergraduate researchers
(Brewster et al., 2004). The research program begun and described here will provide the basis for
the creation of further undergraduate research opportunities at our University (NSU).
Maria Farrell and Emily Schmitt
Nova Southeastern University
Department of Math, Science, and Technology
Ft. Lauderdale, FL
MATERIALS & METHODS - OUTLINE
Protocol Established
•Yeast grown (20 hours) in YEPD to an optical density (OD) of 0.6-0.8 at 660nm
•Total RNA was extracted
•Total RNA quality and quantity documented
- Electrophoresis Gel to visualize ribosomal subunits (3000 and 1800 bp)
- Spectrophotometry to calculate RNA density and find ratio of RNA to DNA (1.8-2.1)
•cDNA made from total RNA using Genisphere Array 350 Protocol
•cDNA presence verified using TDH1 “housekeeping” gene
- PCR to isolate and make copies of TDH1 from the cDNA sample
- Electrophoresis Gel to confirm the fragment was of the appropriate size (206 bp)
•Labeled cDNA hybridized to the microarray
•Microarray slides scanned
•Data Analyzed
Yeast Grown in Experimental Conditions
•Yeast grown in minimal media with 200 mM and 0 mM Al to OD of 0.6-0.8 at 660 nm
•Microarrays prepared following protocol established above
•Dye swap performed
- slide #22 red=200mM Al, green=0 mM Al
- slide #23 red= 0 mM Al, green=200mM Al
•Microarray slides scanned
•Data analyzed: Ten distinct spots compared among all six arrays (3 slides)
DISCUSSION
In this study when yeast were exposed to aluminum, a potentially toxic agent, numerous spots were
clearly green or red indicating differential expression among the two environments. Upon investigation,
many of these spots pertained to mitochondrial enzymes involved in the Krebs cycle, to chaperones
involved in protein folding, refolding, and/or sequestering, and to uncharacterized proteins. One of the
brightest spots on the array for yeast in the presence of aluminum was a gene coding for a possible
chaperone protein, Hsp32. Under conditions of stress native proteins often unfold (Tam, 2002) causing an
increased need for chaperones to refold the proteins “or protect the cell from the potentially toxic affects of
their aggregation” (Wilson, et. al, 2004). In this case Hsp32 has actually been linked to a group of genes
involved in Parkinson’s disease (SGD, 2005). In other cases it is the downregulation of chaperone genes
that is thought to contribute to disease (Tam, 2002). Another gene expressed only in the aluminum
environment was ATG3, a gene that codes for a protein involved in autophagy, a process by which a cell
consumes itself in times of stress. According to Shintani and Klionsky, 2004 autophagy may protect the
cell or cause disease. In some forms of degenerative diseases such as Parkinson’s, autophagic vesicle
concentrations have been found to be elevated. One of the brightest spots on the array for yeast in the
absence of aluminum was ISU2, a gene coding for an Fe/S cluster. Interestingly, Zatta 2000 found that
another Fe/S cluster, aconitase (ACO1), required for mitochondrial function was reduced in expression, as
were others, in the presence of aluminum in rat brains. In our experiment ACO1 was expressed in all three
environments. Finding the repression of ISU2 may be significant for two reasons: aluminum is believed to
be a competitive inhibitor of iron and because researchers have linked alterations in mitochondrial
enzymes such as this to Alzheimer’s disease.
The specific mechanism of aluminum toxicity on an entire genome remains elusive due to the diverse
pathways affected. However, with microarray technology the interactions between thousands of genes of
known and unknown function can be identified and investigated further. This is one advantage to bringing
such a project to the undergraduate curriculum. Students can predict genes that may be affected by various
experimental conditions, use microarray technology to broadly test their predictions, and later verify these
results with other tools such as blots and knock out experiments. By learning to design experiments using
microarrays and analyzing the resulting data students gain valuable skills that will be essential to a broad
range of biological study including medicine and pharmaceutical advancement as well as basic and applied
research (Brewster et al., 2004). We have only begun to analyze the data resulting from this pilot study
and expect to find additional discoveries in our exploration of how the yeast genome is affected by
environmental contaminants.
Table 1: Genes expressed only in 200 mM Al and not expressed in the YEPD (yellow) array.
Named
gene
HSP32
Figure 1: Gel Visualization of TDH1 gene. Lane 1 is a ladder
containing DNA fragments (5000, 4000, 3000, 2500, 1500, 1000,
900, 800, 700, 600, 500, 400, 300, 200, 100 bp). Lane 2 is the
TDH1gene isolated from a representative cDNA sample.
COS111
Figure 2: A grid from the yellow array resulting from two green and red fluorescently tagged
cDNA prepared from the mRNA of yeast growing in similar YEPD cultures. The TDH1 gene is
indicated by a red arrow.
DAL80
GRE1
MATERIALS & METHODS
A protocol was established by performing a control array and intermediate results were verified
using PCR and electrophoresis. The mRNA from two cultures of yeast grown in the same
standard YEPD environment was extracted (Ambion, 2004), converted to cDNA and fluorescently
tagged, one with red Cy3 and the other with green Cy5 dye. The presence of cDNA was verified
(Figure 1) using PCR and electrophoresis of a housekeeping gene, TDH1 (Bradford, et. al, 2005).
The cDNA was hybridized onto a whole genome yeast 70-mer microarray (Genisphere, 2004). As
expected the resulting array was mostly yellow (Figure 2). Microarrays were provided by
Washington University (St. Louis) through the Genome Consortium for Active Teaching (GCAT,
2004). Each slide had two identical arrays printed on it, such that data from two arrays were
obtained from hybridization to one slide. Next, yeast were grown in experimental conditions,
standard YEPD with low amino acids (minimal media) versus minimal media with 200mM
aluminum. A low amino acid agar solution was specifically used to keep the aluminum from
being precipitated out in the presence of phosphates. Using the established protocol, the mRNA
was taken from each condition, converted to cDNA, fluorescently tagged and hybridized to the
microarray slide. A dye swap was preformed.
Slides were scanned with a calibrated GenePix 4000a Microarray Scanner (Axon Instruments)
at wavelengths 532 nanometers and 635 nanometers with PMT settings of 650 and 700
respectively. The 16-bit two channel TIFF array image was analyzed by GenePix Pro 4.1 (Axon
Instruments) software at the University of Miami’s DNA Microarray Core Facility (UM, 2004).
Data was gridded and analyzed using MicroArray Genome Imaging and Clustering Tool (MAGIC
Tool) freely available from the GCAT (Heyer et al., 2005). Genes expressed under the various
conditions (and among dye swaps) were examined and compared between experimental and
control arrays. Five very bright, distinctive spots were selected which were expressed only in the
200 mM Al and not the 0 mM Al environment or the YEPD (control array). The same process was
followed for genes expressed in both the 0 mM Al environment and in the standard YEPD array.
The possible significance of these genes was discussed based on the available primary literature.
ORF
Notes*
YPL280W
Possible chaperone and cysteine protease
with similarity to E. coli Hsp31 and S.
cerevisiae Hsp31p, Hsp33p, and Sno4p;
member of the DJ-1/ThiJ/PfpI
superfamily,which includes human DJ-1
involved in Parkinson’s disease
YBR203W
Protein required for wild-type resistance to
theantifungal drug ciclopirox olamine; not
related to the COS family of subtelomericallyencoded proteins
YKR034W
Negative regulator of genes in multiple
nitrogen
degradation pathways; expression is regulated
by
Nitrogen levels and by Gln3p; member of the
GATAbinding family, forms homodimers and
heterodimerswith Deh1p (Basu et.al, 2004)
YPL223C
Hydrophilin of unknown function; stress
induced
(osmotic,ionic, oxidative, heat shock and
heavy metals); regulated by the HOG
pathway
Autophagocytosis; Protein involved in
autophagy; E2 like enzyme that plays a role in
formation of Atg8p phosphatidylethanolamine
* Notes obtained from the Saccharomyces Genome Database (www.yeastgenome.org)
conjugates, which are involved in membrane
dynamics during autophagy
ATG3
YNR007C
Table 2: Genes expressed only in 0 mM Al and also expressed in the YEPD (yellow array)
Named
gene
PHO5
ISU2
HIS1
GPX2
HCH1
ORF
Notes*
YBR093C
One of three repressible acid phosphatases, a
glycoprotein that is transported to the cell
surface by the secretory pathway; induced by
phosphate starvation and coordinately
regulated
YOR226C
Conserved protein of the mitochondrial matrix,
required for synthesis of mitochondrial and
cytosolic iron-sulfur proteins, performs a
scaffolding function in mitochondria during Fe/S
cluster assembly; isu1 isu2 double mutant is
inviable
YER055C
ATP phosphoribosyltransferase, a hexameric
enzyme, catalyzes the first step in histidine
biosynthesis; mutations cause histidine
auxotrophy and sensitivity to Cu, Co, and Ni
salts; transcription is regulated by general
amino acid control.
YBR244W
Phospholipid hydroperoxide glutathione
peroxidase induced by glucose starvation that
protects cells from phospholipid hydroperoxides
and nonphospholipid peroxides during
oxidative stress
YNL281W
Heat shock protein regulator that binds to
Hsp90p and may stimulate ATPase activity;
originally identified as a high-copy number
suppressor of a HSP90 loss-of-function
mutation; GFP-fusion protein localizes to the
cytoplasm and nucleus.
LITERATURE CITED
Figure 3: Gene expression patterns for yeast exposed to 200 mM Al (green) and 0 mM Al (red) in minimal media (slide # 23b).
RESULTS
The arrays indicate that many genes were induced or repressed in the experimental
environment denoted by very strong signaling of their corresponding florescent labels. (Figure 3).
Qualitative analysis suggests that more genes were expressed in the 0 mM Al environment than in
the 200 mM Al environment. Comparing the arrays on each slide to the dye swap helped to rule
out preferential binding of the dyes, which could lead to inaccurate conclusions regarding gene
expression when in reality there was a difference in dye binding alone. Based on conclusive
signaling quality from all six arrays five genes that were expressed exclusively in the aluminum
environment included HSP32, COS11, DAL80, GRE1, and ATG3 (Table 1). On the other hand,
five genes that were expressed only in the 0 mM aluminum minimal YEPD included PHO5,
ISU2, HIS1, GPX2, and HCH1 (Table 2). In addition to discovery of genes potentially affected
by the presence of aluminum, protocols were established for using microarray technology with
resources available to students in the undergraduate laboratory environment.
Ambion (2004). RiboPure TM Yeast Instruction Manual. Version 0306. Retrieved Sept 2004 from http://www.ambion.com/techlib/prot/fm_1926.pdf
Anoop, V., Basu, U., McCammon, M., McAlister-Henn, L., Taylor, G. (2003). Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial
citrate synthase. Plant Physiology Vol 132, pp. 2205-2217.
Barnett, B. (2002). Toxicity, Aluminum. Retrieved Dec. 3, 2005 from http://www.emedicine.com/med/topic113.htm
Basu, U., Southron, J.L., Stephens, J.L., Taylor, G.J. (2004). Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of PHGPX and URE2 genes in aluminum resistance in
Saccharomyces cerevisiae. Mol Gen Genomics 271; 627-637.
Bradford, W., Cahoon, L., Freel, S., Mays Hoopes, L., Eckdahl, T. (2005). An inexpensive gel electrophoresis-based polymerase chain reaction method for quantifying mRNA levels. Cell Biology
Education, Vol 4;157-168
Brewster, j., Beth Beason, K., Ecktahl, T., Evans, I (2004). The microarry revolution. Biochemistry and Molecular Biology Education. Vol 32, No 4, pp 217-227.
Canada Department of Health (2004). Health Canada. Retrieved from http://www.hc-sc.gc.ca/hesc/water/factsheets/aluminum-human-health.htm
Ezaki, B., Gardner,R., Ezaki, Y., Kondo, H., Matsumoto, H. (1997). Protective roles of two aluminum (Al)- induced genes, HSP150 and SED1 of Saccharomyces cerevisiae, in Al and oxidative stresses.
FEMS Microbiology Letters. 159, 99-105.
Genisphere (2004). 3DNA Array 350 Expression Array Detection for Microarrays. Adapted from version 10-19-04. www.genisphere.com
Genome Consortium for Active Teaching (2004) http://www.bio.davidson.edu/projects/GCAT/gcat.html
Heyer, L.Moskowitz, D., Abele, J., Karnick, P., Choi, D., Campbell, A. Oldham, E. Akin, B., MAGIC Tool: integrated microarray data analysis. Bioinformatics. Vol 21, No 9, pp 2114-2115.
MacDiarmid, C., Gardner, R. (1998). Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion. The Journal of Biological Chemistry Vol 273, No
3, pp. 1727- 1732, Jan 16, 1998
National Human Genome Research Institute-NGHI (1996). 1196 Release Yeast Genome Sequence. Retrieved 01-March from http://www.genome.gov/10000510
Saccharomyces Genome Database (SGD). Retrieved July 28, 2005 from http://www.yeastgenome.org/
Shintani, T, Klionsky, D J (2004). Autophagy in health and disease: a double-edge sword. Science, 306, 990-995.
Tam, S. (2002). Motif finding in upstream regions of chaperone genes downregulated in response to environmental changes. Retrieved Aug 2005 from
http://cmgm.stanford.edu/biochem218/Projects%202002/Tam.pdf
University of Miami Core Facility (UM, 2004). Materials and Methods. Retrieved December 2004 from http://chroma.med.miami.edu/arrays/
Wilson,M., St. Amout,C., Collins,J., Ringe,D.,Petsko,G. (2004). The 1.8-A resolution crystal structure of YDR522Cp from Saccharomyces cerevisae: A member of the DJ-1/ThiJ/PfpI superfamily.
Biophysics. Vol 101, No 6, pp. 1531-1536
Zatta, P., Lain, E., Cagnolini, C. (2000). Effects of aluminum of activity of Krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate. Eur. J. Biochem. 267, pp. 3029-3055.
ACKNOWLEDGMENTS
We thank Ginger Zara of Ambion, Inc., Jessica Bowers of Genisphere, Inc., Mary Lee Ledbetter, Malcolm
Campbell, Todd Eckdahl, and Laura Hoopes of GCAT, Don Rosenblum, Matthew He, Dimitri Giarikos, Emil Kozarov,
Zaki Darojat of Nova Southeastern University (NSU) and many others for their generous assistance. This work was
made possible by a Faculty Development Grant from NSU to E. Schmitt and an Undergraduate Honors Thesis Award
to M. Farrell.